The isolated comet tail pseudopodium of Listeria monocytogenes: a tail of two actin filament populations, long and axial and short and random.

Sechi AS, Wehland J, Small JV - J. Cell Biol. (1997)

Bottom Line:
The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin.We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane.A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.

ABSTRACTListeria monocytogenes is driven through infected host cytoplasm by a comet tail of actin filaments that serves to project the bacterium out of the cell surface, in pseudopodia, to invade neighboring cells. The characteristics of pseudopodia differ according to the infected cell type. In PtK2 cells, they reach a maximum length of approximately 15 microm and can gyrate actively for several minutes before reentering the same or an adjacent cell. In contrast, the pseudopodia of the macrophage cell line DMBM5 can extend to >100 microm in length, with the bacteria at their tips moving at the same speed as when at the head of comet tails in bulk cytoplasm. We have now isolated the pseudopodia from PtK2 cells and macrophages and determined the organization of actin filaments within them. It is shown that they possess a major component of long actin filaments that are more or less splayed out in the region proximal to the bacterium and form a bundle along the remainder of the tail. This axial component of filaments is traversed by variable numbers of short, randomly arranged filaments whose number decays along the length of the pseudopodium. The tapering of the tail is attributed to a grading in length of the long, axial filaments. The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin. We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane. A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.

Figure 2: Characteristics of pseudopodia from PtK2 cells (a and b), macrophages (c and d), and Hela cells (e and f). (a–d) Difference in appearance between Listeria comet tails (arrowheads in a and c) and pseudopodia (a–d) after phalloidin staining. (e and f) Presence of α-actinin in comet tails and absence from pseudopodia. Pseudopodia are clearly identified by phalloidin label (e) but are negative for α-actinin (f). (g and h) A comet tail in the process of forming a pseudopodium and showing the lack of α-actinin in the protruding region. Arrowheads indicate position of rear end of bacterium. (i and j) Ezrin is present in pseudopodia but not in comet tails in the main cell body (the faint label of the tails in the cell body in f is due to some bleedthrough from the rhodamine channel (i). (Inset) Labeling of a pseudopodium that had detached from a cell but was still bound to the coverslip. Bars: (a–d, i, and j) 7.5 μm; (e and f) 12 μm; (g and h) 5 μm; (inset) 1.8 μm.

Mentions:
After phalloidin labeling, it was evident that there was a lower amount of F-actin in the pseudopodia, as compared with the tails in the cell body. The tails in the pseudopodia appeared generally slimmer and, for the longer ones, the phalloidin label tapered down to a thin stalk that connected to the cell periphery (Fig. 2, a–d). This difference in morphology between comet tails in the deeper cytoplasmic and pseudopod compartments correlated with the changes in phase density observed in the phase-contrast microscope. In the bulk cytoplasm, the tails appeared diffuse, whereas in the pseudopods, where they were delimited by the cell membrane, they appeared more phase dense and compact.

Figure 2: Characteristics of pseudopodia from PtK2 cells (a and b), macrophages (c and d), and Hela cells (e and f). (a–d) Difference in appearance between Listeria comet tails (arrowheads in a and c) and pseudopodia (a–d) after phalloidin staining. (e and f) Presence of α-actinin in comet tails and absence from pseudopodia. Pseudopodia are clearly identified by phalloidin label (e) but are negative for α-actinin (f). (g and h) A comet tail in the process of forming a pseudopodium and showing the lack of α-actinin in the protruding region. Arrowheads indicate position of rear end of bacterium. (i and j) Ezrin is present in pseudopodia but not in comet tails in the main cell body (the faint label of the tails in the cell body in f is due to some bleedthrough from the rhodamine channel (i). (Inset) Labeling of a pseudopodium that had detached from a cell but was still bound to the coverslip. Bars: (a–d, i, and j) 7.5 μm; (e and f) 12 μm; (g and h) 5 μm; (inset) 1.8 μm.

Mentions:
After phalloidin labeling, it was evident that there was a lower amount of F-actin in the pseudopodia, as compared with the tails in the cell body. The tails in the pseudopodia appeared generally slimmer and, for the longer ones, the phalloidin label tapered down to a thin stalk that connected to the cell periphery (Fig. 2, a–d). This difference in morphology between comet tails in the deeper cytoplasmic and pseudopod compartments correlated with the changes in phase density observed in the phase-contrast microscope. In the bulk cytoplasm, the tails appeared diffuse, whereas in the pseudopods, where they were delimited by the cell membrane, they appeared more phase dense and compact.

Bottom Line:
The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin.We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane.A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.

ABSTRACTListeria monocytogenes is driven through infected host cytoplasm by a comet tail of actin filaments that serves to project the bacterium out of the cell surface, in pseudopodia, to invade neighboring cells. The characteristics of pseudopodia differ according to the infected cell type. In PtK2 cells, they reach a maximum length of approximately 15 microm and can gyrate actively for several minutes before reentering the same or an adjacent cell. In contrast, the pseudopodia of the macrophage cell line DMBM5 can extend to >100 microm in length, with the bacteria at their tips moving at the same speed as when at the head of comet tails in bulk cytoplasm. We have now isolated the pseudopodia from PtK2 cells and macrophages and determined the organization of actin filaments within them. It is shown that they possess a major component of long actin filaments that are more or less splayed out in the region proximal to the bacterium and form a bundle along the remainder of the tail. This axial component of filaments is traversed by variable numbers of short, randomly arranged filaments whose number decays along the length of the pseudopodium. The tapering of the tail is attributed to a grading in length of the long, axial filaments. The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin. We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane. A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.